Development of a purified cholera toxoid. III. Refinements in purification of toxin and methods for the determination of residual somatic antigen

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Abstract
The addition of an ultrafiltration step to a purification procedure previously described for cholera toxin permitted the preparation of highly purified antigenic toxoids essentially free of somatic antigen(s). The purity of such toxoids is established by the absence of more than .apprx. 1 part Limulus amebocyte lysate (LAL)-positive endotoxin/105 parts toxoid and by the inability of the toxoids to elicit a significant rise in rabbit vibriocidal antibody. The antigenicity of the toxoids is demonstrated by their ability to produce the same high levels of rabbit serum antitoxin as are produced by comparable toxoids containing small amounts of somatic antigen. Amounts of somatic antigen .apprx. .ltoreq. 1 .mu.g/100 .mu.g of toxoid do not exert an adjuvant effect on the toxoid, at least with respect to circulating antitoxin. Where present, the ability of somatic antigen to elicit vibriocidal antibody is influenced by the immunization schedule employed. A correlation exists between the LAL-determined endotoxin content of the toxoids and their ability to stimulate vibriocidal antibody.